Human Respiratory Syncytial Virus (HRSV) is the single largest viral cause of pediatric bronchiolitis and pneumonia. With an estimated mortality of >100,000 children per year worldwide, development of an anti- HRSV vaccine is a priority. Among the current approaches, live-attenuation is attractive because, unlike inactivated vaccines, it promises to induce a broad and balanced immune response. However, live HRSV vaccines have so far been unable to fully prevent potentially dangerous side-effects in infants and children. The long-term objectives of this research are to overcome the safety challenges of live-attenuated HRSV vaccines and to contribute fundamental knowledge of the HRSV life cycle to design alternative anti-HRSV approaches. To meet these objectives, this proposal focusses on molecular manipulation of the viral matrix (M) protein to enhance safety of live-attenuated vaccines. The M protein is essential for replication and plays a prominent role in virion-assembly processes, many of which are highly relevant for the production, composition, release, and perhaps stability of virus particles. A better understanding of M functions therefore offers significant potential for vaccine advancements. A novel system was developed based on an infectious virus lacking the M gene (M-null) which allows rapid screening and manipulation of M functions. By providing plasmids expressing M mutants to cells infected with the M-null virus, a preliminary screen identified M mutations with potential to regulate the level of infectious progeny production of a live virus. This proposal utilizes the M-null based system to likewise identify and manipulate assembly-relevant M functions and test the translational potential in vivo, through the following Specific Aims (abbreviated): 1) Identify M functions and mutations, and the underlying mechanisms, that regulate virus assembly, composition, and release. 2) Determine the quality ofthe immune response to live viruses with transmission-deficiencies based on M mutations, in vitro and in vivo. 3) Test promising M mutant viruses for ability to protect mice after challenge with wildtype HRSV. Together these aims will raise our fundamental understanding of HRSV replication and test the potential of M protein manipulation to contribute to the generation of a safe live-attenuated vaccine.